Group A Streptococcus strains causing meningitis without distinct invasive phenotype

Abstract

Group A streptococcal (GAS; aka Streptococcus pyogenes) meningitis is a fulminant disease associated with high morbidity and mortality. To elucidate the mechanisms underlying the invasiveness of GAS in meningitis, we compared GAS isolates derived from five cases of meningitis to otitis and colonizing isolates. We did not observe differences in adherence to and invasion of human brain microvascular endothelial cells, virulence factors activity, or barrier disruption. Whole genome sequencing did not reveal particular invasiveness traits. Most patients previously suffered from otitis media suggesting that meningitis likely resulted from a continuous spread of the infection rather than being attributable to changes in the pathogen's virulence.

Keywords: Group A Streptococcus; meningitis; virulence determinants.

Figure 1

Patient scans and histology results. (a) CT‐scan: Septic thrombosis with mastoiditis and otitis media on the right side. The red arrow indicates thrombosis. (b) MRI‐Scan: T2 sequence showing penetration of lower temporal lobe with abscess formation. The red arrow indicates an abscess. (c) HE Staining of mastoid tissue: the black arrow indicates necrotic mastoid tissue, and the dashed black arrow indicates mixed neutrocytic and lymphocytic infiltration, foam cells, and cholesterol crystals (not depicted) indicating chronic inflammation. (d) Brown–Brenn staining of mastoid tissue: The arrows indicate the accumulation of extracellular bacteria in chains. CT, computed tomography; H&E, hematoxylin and eosin; MRI, magnetic resonance imaging.

Figure 2

Bacterial strains virulence determinants. (a) Adherence to and (b) invasion of human brain vascular endothelial cells (HBMECs) were assessed after 30 min (MOI 1) or 2 h (MOI 10) of infection, respectively. Adherence and invasion percentages were calculated based on the initial bacterial inoculum used to infect HBMECs. Bacterial adherence above 100% occurred in one case and could be explained by increased initial bacterial growth or by disruption of long bacterial chains or clumps due to sheer pipetting forces during the eukaryotic cell lysis step. Bacterial virulence factors activity of (c) Streptococcal DNases, (d) the IL‐8 protease SpyCEP, and (e) the pore‐forming toxin SLO were assessed in supernatants of exponentially growing GAS. (f) HBMECs barrier function disruption was carried out with the ECIS® Z‐Theta instrument (Applied Biophysics), measuring the impedance generated by the cell barrier in the presence or absence of bacteria. Control experiments were carried out using medium only. (a–d) The average of the biological replicates for each strain is depicted as a single data point on the graphs so that each data point represents a single strain. The two strains indicated in the figure legend with a black symbol (CI407 and CI571) are the only strains of emm‐type 28 (M28), as opposed to all other strains that are emm‐type 1 (M1). (a–e) Three biological replicates were carried out for each strain. The average values for strains from a single group of isolates (meningitis, otitis, or colonizing) are depicted in the graphs and the error bars represent the standard deviation. (f) Two biological replicates were carried out for each strain and the average of the strains from to the same group is depicted on the graph. Control = medium only. No significant differences in adherence, invasion, virulence factors activity, or barrier disruption were observed among the different groups of isolates. A significant difference was found between the respective controls and meningitis, otitis, or colonizing isolates for DNase (p = 0.0159, 0.0159, and 0.0159, respectively) and SpyCEP (p = 0.0179, 0.0357, and 0.0179, respectively) activity. Statistical significance was assessed using the Mann–Whitney test. Col, colonizing; Ctrl, control; Men, meningitis.